CHARACTERIZATION OF A NOVEL PROTEIN-KINASE-C RESPONSE ELEMENT IN THE GLUCAGON GENE

To maintain glucose levels in blood within narrow limits, the synthesi s and secretion of pancreatic islet hormones are controlled by a varie ty of neural, hormonal, and metabolic messengers that act through mult iple signal transduction pathways, Glucagon gene transcription is stim ulated by cyclic AMP and depolarization-induced calcium influx, In thi s study, the effect of protein kinase C on glucagon gene transcription was investigated, After transient transfection of a glucagon-reporter fusion gene into the glucagon-producing islet cell line alpha TC2, ac tivation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate ( TPA) stimulated glucagon gene transcription, By 5' deletions, 3' delet ions, internal deletion, and oligonucleotide cassette insertion, the T PA-responsive element was mapped to the G2 element (from -165 to -200) , Like TPA, overexpression of oncogenic Ras (V-12 Ras) stimulated GZ-m ediated transcription whereas overexpression of a dominant negative Ra s mutant (N-17 Ras) blocked the effect of TPA. A mutational analysis o f G2 function and nuclear protein binding indicated that protein kinas e C and Ras responsiveness is conferred to the glucagon gene by HNF-3 beta functionally interacting with a protein that binds to a closely a ssociated site with sequence similarity to binding sites of Ets family proteins, HNF-3 beta belongs to the winged-helix family of transcript ion factors and has been implicated in the control of cell-specific an d developmental gene expression, The results of the present study show that the cell lineage-specific transcription factor HNF-3 beta is an essential component of a novel protein kinase C response element in th e glucagon gene.